text: The original ICE protocol involved an RT-PCR amplification step and therefore required primers and knowledge of the location or regions to be investigated, alongside a maximum cDNA length of 300–500bp.
  The ICE-seq method is complicated, along with being labour-, reagent- and time-intensive. One protocol from 2015 took 22 days. This shares a limitation with inosine-specific cleavage, in that if there are two A-to-I modifications in relatively close proximity, the downstream mod is less likely to be detected since the cDNA synthesis will be truncated at a prior nucleotide.
  Both ICE and ICE-seq suffer from a lack of sensitivity to infrequently edited locations: it becomes difficult to distinguish a modification with a frequency of <10% from a false positive. An increase in read depth and quality can increase sensitivity, but also then suffer from further amplification bias.